Liposome-based drug carriers can effectively enhance drug efficacy while reducing toxicity, and they have considerable potential as drug delivery platforms in cancer (Drummond, D. C. et al, Pharmacol. Rev. 1999, 51 (4), 691-743; Allen, T. M.; Cullis, P. R., Science 2004, 303 (5665), 1818-22). Examples include liposomal doxorubicin, and liposomal cytarabine, both FDA approved for cancer treatment (Gabizon, A. et al. J Control Release 1998, 53 (1-3), 275-9; Bomgaars, L. et al., J Clin. Oncol. 2004, 22 (19), 3916-21). However, a key and pervasive obstacle is that many clinically promising drug classes are difficult to stably encapsulate within liposomes (Fritze, A. et al., Biochem Biophys Acta 2006, 1758 (10), 1633-40; Haran, G. et al., Biochim Biophys Acta 1993, 1 151 (2), 201-15).
Staurosporine is a pan protein kinase inhibitor with potent anticancer activity in vitro, but clinical use of this compound is precluded by plasma protein binding with rapid clearance, and non-selective toxicity (Gurley L R et al., Staurosporine analysis and its pharmacokinetics in the blood of rats; Los Alamos National Laboratory; Los Alamos, July 1994). These limitations could conceivably be circumvented by liposomal encapsulation and preferential delivery to tumor tissue, but efficiently loading staurosporine or its analogues into liposomes has thus far not been feasible (Yamauchi, M. et al., Biol Pharm. Bull 2005, 28 (7), 1259-64).
Staurosporine avidly targets the PKC family of signaling proteins in addition to other kinases such as PKA and PKG, which play a key role in tumorigenesis (da Rocha, A. B. et al., Oncologist 2002, 7 (1), 17-33; Sato, W. et al., Biochem Biophys Res Commun 1990, 173 (3), 1252-7; Satake, N. et al., Gen Pharmacol 1996, 27 (4), 701-5). Staurosporine treatment has been proposed for glioblastoma, a lethal cancer for which current treatments are of limited benefit and have serious toxicity (Wen, P. Y. et al., N Engl J Med 2008, 359 (5), 492-507; Stupp, R. et al., J Clin Oncol 2007, 25 (26), 4127-36). However, high staurosporine doses would be required to exceed plasma α1-acid glycoprotein (hAGP) binding effects and allow sufficient free drug for antitumor activity (Fuse, E. et al, Cancer Res 1998, 58 (15), 3248-53), This level of dosing would cause unacceptable toxicity from pan kinase inhibition in normal tissues.
A potential solution to the obstacles hindering further development of staurosporine and its analogues is offered by liposomal encapsulation because: first, liposomes offer improved circulation half-life by shielding payload from plasma hAGP proteins, and they slow hepatic-renal clearance due to optimal sizing combined with PEGylation; and secondly, leaky microvasculature at tumors and metastases facilitates preferential delivery of liposomal payload to tumor tissue, a selective effect called enhanced permeability retention (EPR) that significantly bypasses the blood brain barrier (BBB) and which can be enhanced by tumor cell/vessel targeting of the carrier liposomes (Wang, A. Z. et al., Nanoparticle Delivery of Cancer Drugs. Annu Rev Med 2011; Simberg, D. et al., Biomaterials 2009, 30 (23-24), 3926-33).
Various liposomal remote loading methods incorporating chemical and pH gradients have been developed to encapsulate doxorubicin, topotecan and irinotecan (Drummond, D. C. et al., Cancer Res 2006, 66 (6), 3271-7; Sadzuka, Y. et al., J Control Release 2005, 108 (2-3), 453-9). However, staurosporine encapsulation by liposomes has been poor when attempted with these methodologies (Hashimoto, K. et al., Endocrinol Jpn 1976, 23 (3), 243-9; Yarnauchi, M. et al., Int J Pharm 2008, 351 (1-2), 250-8).
In addition, the delivery of drugs to only specific sites within the body is one central goal of targeted drug therapy. The concept depends on the differential expression of certain target structures only in a subpopulation of tissues or cell types of the body. Therefore, many groups have tried to elucidate differences in the expression of cell surface markers among different tissue types or between the healthy and diseased state of a tissue or cell. Targeted drug delivery is an attractive concept in particular for cancer chemotherapy, which usually administers highly toxic substances systemically. It is believed that targeted drug delivery primarily to the tumor cells or at least primarily into the vicinity of the tumor cells would allow to either increase the amount of chemotherapeutic, which can be administered at the same level of systemic toxicity hut with an increased effect at the tumor site, or to decrease the amount of chemotherapeutic, which is administered thus lowering the systemic toxicity while still eliciting the same effect at the tumor site.
Targeting of the tumor vasculature, thus, represents a promising new approach for targeted cancer therapy (Matter (2001) Drug Discov. Today 6: 1005-1024). Vascular targeting agents are designed to deliver cytotoxic, anti-angiogenic, procoagulant, or proapoptotic substances specifically to the vasculature of tumors. The employed ligands recognize structures associated with tumor blood vessels, i.e., proteins expressed by endothelial cells or associated with the extracellular matrix (Thorpe et al., (2003) Cancer Res. 63: 1144-1147; Halin et al. (2001) News Physiol. Sci. 16: 191-194), Targeting of the vasculature has several advantages over targeting of tumor cells (Augustin (1998) Trends. Pharmacol, Sci. 19: 216-222). There are no physiological barriers as endothelial cells are easily accessible to circulating carrier systems and penetration into the tumor is not necessary. Destruction of few capillary endothelial cells affects a large number of tumor cells depending on them. Since all solid tumors are dependent on neovascularization to grow beyond a few millimeters in diameter, this approach should be broadly applicable. Finally, endothelial cells are genetically stable and do not become resistant to the therapy due to mutation, and only remodeling and expanding endothelium in tumors expresses certain markers such as ανβ3 and ανβ5 integrins, that are not expressed by quiescent, healthy, non-tumor vessels. Therefore such markers have been the object of intense investigation as potential anti-tumor targets (see above references).
In addition ανβ3 and ανβ5 integrins are also found on different tumor cells including metastatic melanoma cells (Conforti et al. (1992) Blood 80: 437-446; Gehlsen et al. (1992) Clin, Exp. Metastasis 10: 111-120; Seftor et al. (1999) Cancer Metastasis Rev. 18: 359-375 and Varner & Cheresh (1996) Carr. Opin. Cell. Biol. 8: 724-730; Nande et al., (2001) J. Gene Med. 3: 353-361), Targeting to ανβ3 and ανβ5 integrins can be achieved by RGD-containing peptides, with a variety of linear and cyclic RGD-containing peptides (DeNardo et al. (2000) Cancer Biother. Radiopharm, 15: 71-79; Pasqualini et al, (1997) Nat. Biotechn. 15: 542-546). Several of these RGD-peptides have already been used to deliver radionuclide, proteins, cytotoxic drugs or viral and non-viral carrier systems to integrin-expressing cells in vitro and in vivo (Arab et al. (1998) Science 279: 377-380; Schraa et al. (2002) Int. J. Cancer 102: 469-475; Jansen et al. (2002) Cancer Res. 62: 6146-6151; Erbacher et al. (1999) Gene Ther. 6: 138-145; Müller et al. (2001) Cancer Gene Ther. 8: 107-117 and Wicknam et al. (1997) J. Virol. 170: 8221-8229; Nande et al, (2001) J. Gene Med. 3: 353-361).
Therefore, there is a need in the art for the development of methods of producing liposomal drug compositions, wherein the drug (e.g., staurosporine) is stably and efficiently encapsulated and wherein the liposome is efficiently delivered to the drug's specific site of action (e.g., a tumor). The present subject matter as provided herein cures these and other needs in the art by providing, inter alia, methods of making liposomally encapsulated drugs (e.g., staurosporine) and compositions related thereto.